Project 1: Structural basis of entry, neutralization, and replication of pandemic influenza viruses Hemagglutinins (HA) are classified into subtypes based on their different antigenic properties. Within viruses of human origin, only HI, H3 and H5 have been structurally analyzed. We propose to determine HA crystal structures from pandemic 1957 H2, as well as recent H7 and H9 human subtypes. Our goal is to identify conserved epitopes in human HAs that could elicit cross- neutralizing antibodies. Antibodies isolated recently from 1918 pandemic survivors have provided invaluable information on neutralization of a highly pathogenic virus. Recent H5 antibodies appear to recognize conserved epitopes on the HA, even across subtj^jes. Understanding the nature of the highly conserved epitopes that can elicit broadly neutralizing antibodies will be of enormous value for rational design of a flu vaccine that can recognize different strains and subt3T3es. To replicate efficiently in humans, zoonotic viruses must engage a host cell receptor to enter and fuse with an internal host cell membrane. HA specificity is correlated with either alpha2,3 sialic acid (avian) or alpha2,6 (human) receptors, the latter required for human-human transmission. Although several HA residues have been implicated in receptor specificity switching, we propose a more comprehensive study to aid surveillance and identify which emerging influenza viruses have the greatest potential to infect and transmit in humans. Replication is highly dependent on the the heterotrimeric RNA-dependent RNA-polymerase from influenza A. The RNA polymerase is an essential pathogenicity factor in the 1918 'Spanish' influenza and highly pathogenic avian H5N1, and mutations affect host specificity. Structural characterization of the polymerase will shed light on the mechanisms which enhance pathogenicity and alter host specificity, as well as providing essential information on viral replication. We also aim to study the pro-apoptotic factor PB1-F2 and its interaction with the host cell. RELEVANCE (See instructions): Pandemic influenza is a constant global threat. Identification of the key features and properties of influenza viruses, that can infect humans is invaluable for the ability to predict and treat future outbreaks. A vaccine that could raise an immune response against multiple subtypes and strains of influenza would be a major advance along with greater understanding of the corrrelates of protection, transmission and replication .